To accelerate progress toward understanding the multiple steps in cancer development, and the fundamental nature of the process of promotion, it is desirable to develop suitable systems for study of these processes in cell culture. This is particularly important in the case of the explicit study of human cancer development, where "whole animal" experimentation is impossible. Previous studies on this project have shown that human endometrial stromal cells repetitively treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) undergo a series of phenotypic alterations which suggest progression toward malignant transformation and resemble several characteristics of human endometrial stromal cell tumors. Furthermore, other studies have demonstrated that both TPA, a strong prototype promoter, and DES, a strong synthetic estrogen, produced effects in cultured endometrial stromal cells that resembled the process of tumor promotion. In both cases the promoter selectively potentiated carcinogen-treated cells and impeded normal cells in their growth and progression toward malignancy. These results lead us to hypothesize that selection may be a manifestation of the fundamental process in tumor promotion. We propose to test this hypothesis through further investigations using an improved, clonally-derived and quantitative system for studying transformation and promotion in human endometrial stromal cells. TPA and DES will be further studied to characterize time and dose parameters of their promoting activity. Other compounds, including natural estrogens of varying potency, phorbol derivatives of varying promoting activity in vivo, and incomplete promoters, will be evaluated in this system to determine whether the phenomenology of selection activity in vitro corresponds to promoting activity in vivo. We will compare whether intentional selection of cells using techniques of cell sorting or replica plating can duplicate the passive selection process in mass culture. Further studies will examine whether expression of oncogenes is altered during promotion in vitro and whether oncogenes transfected into endometrial cells at various stages of progression can induce further changes in these cells. Finally, we will examine several anti-promoters to see whether these compounds have a selective effect inhibiting progressive changes induced during promotion in initiated cells.